Cell death can be assayed by quantifying plasma membrane damage or rupture. The LDH Cytotoxicity Detection Kit offers a simple way to measure plasma membrane damage, based on the release of lactate dehydrogenase (LDH), a stable cytoplasmic enzyme present in most cells.

Highly Sensitive & Accurate

The LDH Cytotoxicity Detection Kit is a simple and accurate colorimetric assay for dead and plasma membrane-damaged cells. LDH present in the culture supernatant (due to plasma membrane damage) participates in a coupled reaction which converts a yellow tetrazolium salt into a red, formazan-class dye which is measured by absorbance at 492 nm. The amount of formazan is directly proportional to the amount of LDH in the culture, which is in turn directly proportional to the number of dead or damaged cells (Figure 1 - Measuring cell death). The assay is extremely sensitive: as few as 2,000 dead or damaged cells per well can be detected (data not shown). Results of LDH and [51Cr] assays for cell-mediated cytotoxicity correlate strongly with each other (1, 2).


Figure 1. Cell death in the presence of 1% Triton X-100 is proportional to the number of cells in the well. K562 cells were titrated in 96-well plates at the indicated cell concentrations. The reaction mixture was added to confirm that the spontaneous release of LDH is low. In the presence of the reaction mixture plus Triton X-100 (final concentration 1%), LDH release increased proportionally to cell concentration, based on measurements of formazan absorbance at 492 nm.

Simple Procedure, Fast Results

The LDH assay does not require prelabeling or washing steps. The entire procedure, from cell growth to measuring results, can be performed in a single 96-well plate. It takes less than an hour once your cells are cultured: Simply add the reaction mixture, incubate for 30 minutes, and measure the absorbance at 492 nm with a multiwell plate reader. Additionally, the assay is nonradioactive for improved safety and simplified cleanup and waste disposal.

In Vitro Applications

LDH assays have been used to quantify cell-mediated cytotoxicity induced by cytotoxic T cells, natural killer cells, lymphokine-activated killer cells, and monocytes (Figure 2 - Measuring cell mediated cytotoxicity; 1, 2), as well as to identify mediators that induce cytolysis (2). These assays have also been used to determine the cytotoxic potential of compounds in environmental and medical research and in food, cosmetic, and pharmaceutical manufacturing (3-9), and to detect cell death in bioreactors (10, 11).

Figure 2. Measuring the cytolytic activity of allogen-stimulated, cytotoxic T lymphocytes. Spleen cells of C57/Bl 6 mice (H-2b) were stimulated in vitro with P815 cells (H-2d). Viable cytotoxic T lymphocytes (CTLs) were purified by ficoll density gradient, washed and titrated in a 96-well plate. 1 ¡¿ 104 P815 test cells/well were added to the effector CTL cells. The cell mixture was centrifuged and incubated for 4 hr. 100 ¥ìl of culture supernatant was collected to measure LDH activity. Panel A. Absorbance values for the effector cell control (orange), effector-test cell mix (blue), and effector-test cell mix minus effector cell control (red). Panel B. Percent cell-mediated cytotoxicity.

Applications
Components Storage Conditions
Catalyst (lyophilized) -20¡ÆC
Dye solution 4¡ÆC for dissolved catalyst and thawed dye solution
User Manual (PT3947-1)


References
  1. Decker, T. & Lohmann-Matthes M.L. (1988) J. Immunol. Methods. 115(1):61-69.
  2. Korzeniewski, C. & Callewaert, D.M. (1983) J. Immunol. Methods. 64(3):313-320.
  3. Dubar, V., et al. (1993) Exp. Lung Res. 19(3):345-359.
  4. Kondo, T., et al. (1993) Toxicol. in Vitro 7(1):61-67.
  5. Murphy, E.J., et al. (1993) Neuroscience 55(2):597-605.
  6. Shrivastava, R., et al. (1992) Cell Biol. Toxicol. 8(2):157-170.
  7. Gelderblom, W.C., et al. (1993) Food Chem. Toxicol. 31(6):407-414.
  8. Thomas, J.P., et al. (1993) J. Lipid Res. 34(3):479-490.
  9. Sasaki, T., et al. (1992) Toxicol. in Vitro. 6(5):451-457.
  10. Legrand, C., et al. (1992) J. Biotechnol. 25(3):231-243. Erratum in: J. Biotechnol. (1993) 31(2):234.
  11. Racher, A.J., et al. (1990) Cytotechnol. 3 (3):301-307.